Compositions and Methods for Treating Cocaine-Related Disorders

ABSTRACT

A method of treating a cocaine-related disorder in an individual is provided, the method including administering to the individual a therapeutic amount of an antibody comprising a human immunoglobulin gamma heavy chain and a murine lambda light chain. In a specific embodiment, the antibody is a monoclonal antibody comprising a murine lambda light chain variable region, a human gamma heavy chain variable region, and a human kappa or lambda light chain constant region.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.11/788,808, filed Apr. 20, 2007, and claims the benefit of U.S.Provisional Application No. 60/793,604, filed Apr. 20, 2006.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions relatingto monoclonal antibodies.

BACKGROUND OF THE INVENTION

Cocaine is a powerfully addictive stimulant that directly affects thebrain. Cocaine, however, is not a new drug. In fact, it is one of theoldest known drugs. The pure chemical, cocaine hydrochloride, has beenan abused substance for more than 100 years, and coca leaves, the sourceof cocaine, have been ingested for thousands of years.

Today, cocaine use ranges from occasional use to repeated or compulsiveuse, with a variety of patterns between these extremes. There is no safeway to use cocaine and any route of administration can lead toabsorption of toxic amounts of cocaine, leading to acute cardiovascularor cerebrovascular emergencies that could result in sudden death.Repeated cocaine use by any route of administration can producedependence, addiction and other adverse health consequences.

Despite decades of basic and clinical research there are currently nomedications available to treat cocaine dependence, addiction, overdoseor to help prevent relapse. Thus, therapies are needed which can treatsuch cocaine-related disorders.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed toward a method fortreating a cocaine-related disorder in an individual. The methodincludes administering to the individual a therapeutic amount of anantibody comprising a human gamma heavy chain and a murine lamda lightchain.

In another embodiment, the present invention is directed toward a methodfor treating a cocaine-related disorder in an individual. The methodcomprises administering a therapeutic amount of an antibody comprising ahuman gamma heavy chain and a human kappa light chain at least partiallyderived from 1B3.

In another embodiment, the present invention is directed toward amonoclonal antibody comprising a human gamma heavy chain and a murinelambda light chain.

In another embodiment, the present invention is directed toward amonoclonal antibody comprising a human gamma heavy chain and a humankappa light chain at least partially derived from 1B3.

In an additional embodiment, the present invention is directed toward amethod for binding cocaine or a derivative thereof. The method includescontacting cocaine or a derivative thereof with an effective amount ofan antibody, wherein the antibody comprises a human gamma heavy gammachain and a light chain, wherein the light chain is selected from thegroup consisting of: a murine lambda light chain, a human kappa lightchain derived at least partially from 1B3, and combinations thereof.

These an additional embodiments of the invention will be more fullyapparent in view of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description will be more fully understood in viewof the drawings in which:

FIG. 1 is a graph which depicts pharmacokinetics of an anti-cocaine mAb2E2 where mice receive an i.v. infusion of 120 mg/kg of 2E2, samples ofblood (10 μl) are obtained from tail veins at the indicated times afterthe completion of the mAb infusion, and concentrations of 2E2 in bloodare determined using an ELISA; data points represent the mean±SEM from 8mice, the Vdss is approximately 0.28 l/kg and a single compartment modelwith a t_(1/2) of 8.1 days adequately describes the elimination phase,represented by the best-fit regression line through the data points;

FIG. 2 is a graph which depicts the dose-dependent effect of 2E2 onplasma (A) and brain (B) concentrations of cocaine where mice areinjected with vehicle or 2E2 at doses of 12, 24, 40, 120 or 360 mg/kg;one hour after the infusion of vehicle or 2E2 is completed an i.v. bolusof cocaine HCl is administered and after five minutes the samples arecollected, cocaine concentrations are measured using GC/MS, symbolsrepresent the mean±SEM from three mice, the line through the data pointsrepresents the best fit according to a hyperbolic function, and theED₅₀s of 2E2 for decreasing the cocaine concentration in the brain andincreasing the plasma cocaine concentration are approximately 50 and 60mg/kg, respectively;

FIG. 3 is a graph which depicts the effect of 2E2 on plasma and brainconcentrations of cocaine metabolites where the concentrations of BE andEME are measured in the same tissue samples used for FIG. 2; plasma andbrain concentrations of BE are represented by closed and open squares,respectively, and plasma and brain concentrations of EME are representedby closed and open triangles, respectively; and

FIG. 4 is a graph which depicts the effect of 2E2 on thepharmacokinetics of cocaine in plasma (A) and brain (B) where micereceive an i.v. infusion of 120 mg/kg of 2E2, one hour later the micereceive an i.v. injection of cocaine HCl (0.56 mg/kg), and the animalsare sacrificed at the indicated times and samples are collected; thecocaine concentrations are determined by GC/MS, the data pointsrepresent the mean±SEM from three mice and in the absence of 2E2 (opencircles), the cocaine concentration-time profile in plasma (A) isdescribed by a two-compartment pharmacokinetic model with a t_(1/2α) of1.9 min and a t_(1/2β) of 26.1 min, while in the presence of 2E2 (closedcircles) a single compartment pharmacokinetic model indicated a t_(1/2)of 17.1 min, in the brain (B) in the absence of 2E2 (open circles) atwo-compartment pharmacokinetic model with first order input into thefirst compartment describes the cocaine concentration-time profile andthe calculated input t_(1/2) is 2.0 min and the t_(1/2α) and t_(1/2β)values are 2.0 min and 14.5 min, respectively, and in the presence of2E2 (closed circles), a single compartment pharmacokinetic model withfirst order elimination of cocaine indicates an elimination t_(1/2)value of 3.8 min.

The embodiments set forth in the drawings are illustrative in nature andare not intended to be limiting of the invention defined by the claims.Moreover, individual features of the drawings and the invention will bemore fully apparent and understood in view of the detailed description.

DETAILED DESCRIPTION

As used herein, “cocaine-related disorders” include cocaine dependence,addition, overdose and/or relapse, and any other disorder resulting inwhole or in part from cocaine use. As the site of action of cocaine isin the brain, decreasing the concentrations reaching the brain would beexpected to decrease the probability of dependence, addiction, overdose,and relapse. Antibodies with high affinity and specificity for cocainewould be expected to act as pharmacokinetic antagonists by sequesteringcocaine in the peripheral circulation and preventing its entry to thebrain. Indeed, active immunization of animals with hapten-carrierconjugates can elicit the production of polyclonal anti-cocaineantibodies with sufficient levels and affinity for cocaine that they canreduce the amount of cocaine entering the brain. Active immunization hasalso been shown to attenuate the behavioral effects and the primingeffect of systemically administered cocaine in rats. Furthermore, theability of active immunization to produce levels of polyclonalanti-cocaine antibodies in humans that were associated with a decreasein use of cocaine demonstrates the potential efficacy of immunotherapyfor cocaine abuse. Unfortunately, individuals with compromised immunesystems (like those who have clinically induced immunosuppression orthose who suffer from some sort of an infection) can often not beactively immunized due to the risks of developing a complication fromthe active immunization. Often, those individuals who are suffering fromcocaine-related disorders also have compromised immune systems.

An alternative to active immunization is passive immunization. Inpassive immunization, a pre-made antibody is given to the individual.While this process is usually short lasting (a few days or even a fewweeks), it is much safer and effective for those with compromised immunesystems. In addition, using a monoclonal antibody (mAb) with a definedaffinity, specificity and dose may be even more efficacious than activeimmunization. Indeed, passive immunization with non-human anti-cocainemAbs attenuates the behavioral effects of cocaine and thereforerepresents an alternative or adjunct to active immunization.

Previously, a murine anti-cocaine antibody (GNC92H2) was generated anddemonstrated to have in vivo efficacy in rat models of cocaineaddiction. Also, two catalytic murine anti-cocaine mAbs that aredesigned to reduce blood cocaine levels through its hydrolysis have beengenerated and characterized. Unfortunately, non-human sequenceanti-cocaine mAbs would be expected to elicit an immune response inhumans similar to that elicited by the murine mAb OKT-3 used forimmunosuppression for organ transplant procedures. This immune responsewill target and try to destroy the non-human mAB thus decreasing orneutralizing the long-term efficacy of such an immunotherapeutic agent.Furthermore, the antibody affinity for cocaine should also be a majordeterminant of clinical efficacy. Unfortunately, the affinities of thecatalytic mAbs for cocaine are reported to be approximately 220 μM and55-5,240 μM, while the affinity of the anti-cocaine mAb GNC92H2 isreported to be 200 nM. Therefore, a more efficacious and predominantlyhuman antibody is likely to decrease the probability of inducing aneutralizing immune system response. This theory led to the generationand characterization of a monoclonal antibodies which are at leastpartially generated in transgenic mice that produce human sequence mAbs.

In contrast to the affinities of the catalytic and non-human mABs, theaffinity of these new antibodies for cocaine is approximately 4 nM,which is considerably higher than that of other anti-cocaine mAbscurrently under study. Additionally, these antibodies have highspecificity for cocaine over the major metabolites of cocaine.Therefore, these antibodies have important physicochemical propertiesthat confer efficacy as a passive immunotherapeutic agent.

Therefore, according to one embodiment, the present invention isdirected toward a monoclonal antibody comprising a human gamma heavychain and a murine lamda light chain. In one embodiment, the murinelambda light chain comprises SEQ ID NO: 1 or a derivative thereof. Inanother embodiment, the human gamma heavy chain comprises SEQ ID NO: 2.In a further embodiment, the human gamma heavy chain comprises SEQ IDNO: 2 and the murine lambda light chain comprises SEQ ID NO: 1. In oneembodiment, the combination of these sequences is known as 2E2. Inanother embodiment, the antibody comprises a human gamma heavy chain anda human kappa light chain derived at least partially from 1B3. Inanother embodiment, the antibody is an immunoglobulin.

According to another embodiment, the invention is directed toward amethod of treating a cocaine-related disorder in an individual. Themethod includes administering a therapeutic amount of an antibody to theindividual including a human immunoglobulin gamma heavy chain and amurine lambda light chain. The human gamma heavy chain contains themajority of the specificity for cocaine and its derivatives and thus itmay be used in combination with any light chain which effects atherapeutic effect for cocaine-related disorders.

In one embodiment, the human gamma heavy chain comprises SEQ ID NO: 2 ora derivative thereof. In an additional embodiment, the light chaincomprises a murine lambda light chain including SEQ ID NO: 1 or aderivative thereof. In one embodiment, the murine lambda light chain ispartially murine derived. In further embodiment, the partially murinederived light chain comprises a murine variable region and a humanconstant region. Derivatives as employed herein include those sequenceswhich would be functionally equivalent to either chain of the antibody.This would include those antibodies which have the same primarystructure (i.e. sequence), but have a different tertiary structure dueto the addition of, for example, a salt or a sugar. In addition, thefunctional regions of the sequences have been identified as the variableregions. Thus, heavy and/or light chains including at least one variableregion and maintaining their ability to be therapeutic forcocaine-related disorders are also included. In the murine lambda lightchain, there are three variable regions. These regions reside at aminoacid residues numbered 23-36 (SEQ ID NO: 4), 52-58 (SEQ ID NO: 5), and91-99 (SEQ ID NO: 6). In the human gamma heavy chain, these regionsreside at amino acid residues numbered 32-37 (SEQ ID NO: 7), 51-67 (SEQID NO: 8), and 100-103 (SEQ ID NO: 9). In the human kappa light chain,these regions reside at amino acid residues numbered 24-34 (SEQ ID NO:10), 50-56 (SEQ ID NO: 11), and 89-98 (SEQ ID NOS: 12 and 13).

The variable regions or CDR regions are the sites for ligand-antibodyinteractions. Thus, in one embodiment, derivatives of SEQ ID NO: 1 maycomprise one or more of SEQ ID NOS: 4, 5, and 6 and derivatives of SEQID NO: 2 may comprise one or more of SEQ ID NOS: 7, 8, and 9.Additionally, derivatives of the variable regions could also be used,for example derivatives having between 90-95%, 85-90%, 80-85%, 75-80%,70-75%, 65-70%, or 60-65% homology to their respective variable regionsequences. The resulting antibodies from the derivatives of the variableregions also have the same cocaine binding functionality as the otherdisclosed antibodies.

In a another embodiment, a monoclonal antibody that specifically bindscocaine is provided, wherein the antibody comprises a) a murine lambdalight chain variable region complementarity determining region 1 (CDR1)comprising SEQ ID NO: 4; (b) a murine lambda light chain variable regionCDR2 comprising SEQ ID NO: 5; (c) a murine lambda light chain variableregion CDR3 comprising SEQ ID NO: 6; (d) a human gamma heavy chainvariable region CDR1 comprising SEQ ID NO: 7; (e) a human gamma heavychain variable region CDR2 comprising SEQ ID NO: 8; (f) a human gammaheavy chain variable region CDR3 comprising SEQ ID NO: 9; and (g) ahuman light chain constant region.

In a specific embodiment, the human light chain constant region isselected from the group consisting of a human kappa light chain constantregion and a human lambda light chain constant region. In a morespecific embodiment, the human light chain constant region is a humanlambda light chain constant region comprising SEQ ID NO: 22.

In an additional embodiment, a method for treating cocaine-relateddisorders includes administering a therapeutic amount of an antibodycomprising a human immunoglobulin gamma heavy chain and a human kappalight chain at least partially derived from 1B3 to an individual. Thislight chain is selected because this sequence has been shown bysequencing of genomic DNA to be generated by light chain generecombination in the hybridoma cell line 2E2 that produces mAb 2E2. Theanti-digoxin mAb 1B3 is a fully human sequence mAb that binds digoxinand digitoxin with high affinity (nMolar). This antibody has beendescribed in two publications. Farr, C. et al., “Three-dimensionalQuantitative Structure-Activity Relationship Analysis of Human SequenceAntidigoxin mAbs using CoMFA.” Journal Medicinal Chemistry 45 (15):3257-3270, 2002 and Paul, S., Monson, N. & Ball, W. J., “MolecularModeling of Cardiac Glycoside Binding by the Human sequence mAb 1B3.”Proteins: Structure, Function and Bioinform. 60: 382-391, 2005,incorporated herein by reference. In one embodiment, the human lightkappa chain comprises SEQ ID NO: 3 (1B3a) or a derivative thereof. Inanother embodiment, the human kappa light chain comprises SEQ ID NO: 14(1B3b) or a derivative thereof. In one embodiment, derivatives of SEQ IDNOS: 3 and 14 may comprise one or more of SEQ ID NOS: 10-13.Additionally, derivatives of their CDR regions are also applicable asdescribed above for SEQ ID NOS: 1 and 2.

In another embodiment, an antibody binds cocaine or a derivativethereof. Radioligand binding assays using an antibody and [³H]-cocaineyields an average dissociation constant (K_(d)) of 4.4 nM. Bindinginhibition constants (K_(i)) of the cocaine derivatives are determinedby competition assays with a constant [³H]-cocaine concentration andvarying concentrations of nonradioactive competitors. The results showthat the present antibodies bind cocaethylene with an affinity(K_(i)=3.4 nM) nearly identical to that for cocaine. On the other hand,the affinities for the three physiologically important but inactivecocaine derivatives benzoylecgonine, ecgonine methyl ester, andecogonine are significantly lower (K_(i) values of 43 nM, 5.2 μM and 95μM, respectively), revealing the importance of the benzoyl moiety forhigh binding affinity. Even K_(d) values of 40 nM show beneficialresults. Other examples of cocaine derivatives include: (−) cocaine;cocaine propyl ester; RTI-128; RTI-66; RTI-160; RTI-192;m-hydroxycocaine; WIN 35, 065-2; WIN 35,428; RTI-31; RTI-32; RTI-55;RTI-111; m-hydroxybenzoylecgonine; p-hydroxybenzoylecgonine; RTI-113;tropine; benztropine; 4′,4″-difluoro-3α-diphenylmethoxytropane;hyoscyamine-N-oxide; methylanisotropine; tropisetronmethiodide;anisodine; scopotamine; scopotamine-N-oxide; methylscopolamine;N-butylscopolamine; (−) pseudococaine; (+) cocaine; norcaine;benzoylnorecgonine; (+) pseudococaine; ecgonidine;exo-6-hydroxytropinone; and methylcocaethylene.

The monoclonal antibodies of the present invention use their bindingaffinity for cocaine and its derivatives to reduce the concentration ofcocaine or its derivatives in the brain. Infused antibodies also producea dramatic dose-dependent increase in plasma cocaine concentrations anda concomitant decrease in the brain cocaine concentrations produced byan i.v. injection of cocaine HCl (0.56 mg/kg). At the highest dose ofantibody tested (3:1, mAb:drug), cocaine is not detectable in the brain.Pharmacokinetic studies show that the normal disappearance of cocainefrom plasma is described by a 2-compartment pharmacokinetic model withdistribution t_(1/2α) and terminal elimination t_(1/2β) values of 1.9and 26.1 min, respectively. In the presence of an equimolar dose of mAb2E2 there is a 26-fold increase in the area under the plasma cocaineconcentration-time curve (AUC) relative to the AUC in the absence of2E2. Consequently, the antibodies of the present invention decreasecocaine's volume of distribution from 6.0 l/kg to 0.20 l/kg, whichapproximates that of 2E2 (0.28 l/kg). However, cocaine is still rapidlycleared from plasma and its elimination is now described by a singlecompartment model with an elimination t_(1/2) of 17 min. Importantly,the antibodies also produce a 4.5-fold (78%) decrease in the cocaine AUCin the brain. Therefore, the effect of the antibodies on plasma andbrain cocaine concentrations is predominantly due to a change in thedistribution of cocaine with negligible effects on its rate ofclearance.

In addition to being a monotherapy, embodiments also include additionalco-therapies. For example, when treating someone in rehabilitation toprevent relapse, an antibody according to the invention can beadministered in conjunction with treatments for withdrawal symptoms (forexample, administration of amantadine and propranolol). An antibodyaccording to the invention can be used in conjunction with counselingand other forms of psychotherapy. In addition, it can be used with anyantagonist or agonist pharmacotherapies that use compounds for which theantibody does not have substantial affinity. The antibody can also beused with other antibodies that target other drugs of abuse ormedicaments.

An antibody according to the present invention may be administered byany suitable route or device. In one embodiment, the antagonist will beadministered by injection. The most common form of delivery will be anintravenous injection or infusion.

The antibody is administered in an amount sufficient to treat thecocaine-related disorder. The treatment as used herein encompasses areduction in clinical symptoms of the disorder and/or elimination of thedisorder. Therapeutic amounts will vary based on an individual's age,body weight, symptoms, and the like, and may be determined by one ofskill in the art in view of the present disclosure. Initial clinicalstudies of a cocaine vaccine do provide vital information about theconcentrations of anti-cocaine antibodies required to decrease cocaineuse by cocaine abusers. In vaccinated patients the highest mean serumantibody titer would correspond to about 61.4 μg/mL, and a decrease incocaine use is reported in this cohort as well as cohorts with lowermean antibody titers. As the standard blood volume in a 70 kg person is2.8 liters, then the quantity of anti-cocaine antibodies that conferefficacy in patients is about 61.4 μg/mL×2,800 ml or about 172 mg/person(about 2.5 mg/kg). This is likely be a minimally effective dose. Bycomparison, doses of 40 and 120 mg/kg can be safely administered and areefficacious in rodent models. These doses translate to 2,800-8,400 mg ina 70 kg person, almost 20-50-fold higher than may be required to conferefficacy in humans. Additionally, it should be noted that the polyclonalanti-cocaine antibodies that constituted the standard immune response invaccinated patients had an average affinity (Kd) of 28 nM [17], whilethe present anti-cocaine monoclonal antibodies have a higher affinity(for example, 2E2 has a Kd=4 nM). Therefore, equimolar doses of 2E2 aremore effective or equieffective doses would be lower.

In addition to its use as a treatment for cocaine-related disorders, theantibodies of the current invention could also be used in screeningassays for the development of other therapeutics, including othertherapeutics useful for the treatment of cocaine. Thus, one embodimentof the present invention also includes a method for binding cocaine or aderivative thereof. The method comprises contacting cocaine or aderivative thereof with an effective amount of an antibody. The antibodycomprises a human gamma heavy chain and a light chain. In oneembodiment, the light chain is selected from the group consisting of: amurine lambda light chain, a human kappa light chain at least partiallyderived from 1B3, and combinations thereof. Additionally, as describedabove, the heavy and light chains can be several different combinationsand/or variations.

The following examples demonstrate various specific embodiments of theinvention.

EXAMPLES Example 1 Methods

Animals.

Jugular vein catheterized male Swiss-Webster mice (22-28 g at the startof the studies) are purchased. Mice are housed individually with freeaccess to food and water and kept on a 12 h light/dark cycle. Thesestudies are carried out in accordance with the Guide for the Care andUse of Laboratory Animals under a protocol approved by the InstitutionalAnimal Care and Use Committee at the College of Medicine, University ofCincinnati.

Cocaine Pharmacokinetic Studies.

Prior to the start of the studies, the patency of the venous cathetersis verified by demonstrating the ability to withdraw blood or injectnormal saline via the catheter. The antibody (3-5 mg/ml) inphosphate-buffered saline (PBS) or vehicle (PBS) is infused at a rate ofapproximately 0.35 ml/min for up to two min, depending on the antibodyconcentration and the body weight of the animal, with the animal heldunder mild restraint. One hour after completion of the infusion of mAb,cocaine HCl (0.56 mg/kg) plus heparin (400 units/kg) is injectedintravenously through the same catheter at a volume of 4.0 ml/kg bodyweight. At most sampling times, sodium pentobarbital (45 mg/kg, i.p) isadministered three minutes prior to sacrificing the animal. For the 0.75min time point the cocaine is injected into anesthetized mice. At thedesignated times after the injection of cocaine, anesthetized mice aresacrificed by decapitation and trunk blood (typically 0.8-1.2 ml) iscollected in a 1.5 ml polypropylene microcentrifuge tube containing 11.2μl heparin (1.0 unit/μl) and NaF (16 mg/0.8 ml of blood) to inhibit,respectively, blood coagulation and enzymatic hydrolysis of cocaine. Theblood samples are centrifuged at 5,000×g for 3 min, then the plasma(typically 0.4-0.8 ml) is carefully separated from packed red bloodcells, placed into sterile 1.5 ml Eppendorf microcentrifuge tubes,rapidly frozen on dry ice and then stored at −80° C. until analysis.

At the same time a separate sample of blood (approximately 100 μl) iscollected from each mouse and rapidly frozen on dry ice then stored at−80° C. The concentration of hemoglobin and, where appropriate 2E2, ismeasured in these samples.

The whole brain is quickly removed from the decapitated mice, surfaceblood is blotted away, and the brain is placed in a polypropylene tube,rapidly frozen on dry ice and then stored at −80° C. until analysis. Foranalysis, brains are weighed and cold deionized, distilled water addedto produce a total volume of 1 ml, then homogenized and centrifuged at13,000 rpm for 45 min at 4° C. The resulting supernatants (0.4-0.6 ml)are collected into sterile polypropylene microcentrifuge tubes and analiquot (0.05-0.40 ml) is processed for cocaine/metabolite analysis byGC/MS and hemoglobin content. Any remaining sample is stored at −80° C.

Determination of Blood and Brain Hemoglobin Concentrations.

The hemoglobin contents of brain and blood are quantifiedspectroscopically by combining the method reported by Choudhri et al.(1997) and a protocol provided by Pointe Scientific, Inc. (MI). In thisprocedure, 10 μl aliquots of blood or 50 μl aliquots of brain homogenatesupernatants are diluted with 90 μl hemoglobin reagent (0.6 mMK₃Fe(CN)₆, 0.7 mM KCN) in glass test tubes. The reaction is allowed toproceed at room temperature for 15 min with gentle mixing. When thereaction is complete, aliquots from the standards and samples are alltransferred into PVC microtiter plate wells and the absorbance ismeasured at a wavelength of 490 nm for the measurement ofcyanmethemoglobin formation. For the similarly prepared hemoglobinstandards, the absorbance is directly proportional to the hemoglobinconcentration over the range used (0.3-12 g/dl). The standard curve isverified using control standards and the hemoglobin concentration ineach sample is determined by comparison with the standard curve. Themean±SEM concentration of hemoglobin in whole blood and brain aredetermined to be 8.90±0.32 g/dl and 0.22±0.04 g/dl, respectively. Theaverage hemoglobin content in brain tissue relative to that present inwhole blood is, therefore, approximately 2.5%.

2E2 In Vivo Pharmacokinetic Studies: Sample Preparation.

Mice, while under mild restraint, are administered mAb 2E2 (120 mg/kg,at 4.2 mg/ml in PBS) via an intravenous infusion over a 2 min period.Then at varying times, to obtain blood samples for mAb quantification,the mice are anesthetized using isoflurane and a sterile 27-gaugehypodermic needle or, alternatively, a sterile scalpel blade is used topuncture or make a small cut in a tail vein and 10 μl of blood iscollected using a heparinized capillary pipette tip. The blood isimmediately placed in a 1.5 ml polypropylene microcentrifuge tubecontaining 40 μl of 0.1 M sodium citrate/0.1% sodium azide pH 4.75.These samples are then rapidly placed on ice and then stored at 4° C.until use. A blood sample is taken immediately prior to the infusion of2E2 and then at 3, 15 and 30 min, 1, 2, 4 and 8 hr, 1 day andperiodically up to 49 days as shown in the results.

mAb 2E2 Quantification: ELISA.

The in vivo concentrations of 2E2 are determined using an enzyme-linkedimmunosorbent assay (ELISA) that compares the quantity of mAb in varyingdilutions of the mouse blood samples to that quantified in a standardcurve generated using known dilutions of purified 2E2 or human IgG.Briefly, the conjugate benzoylecgonine-ovalbumin (3 μg/ml, 100 μl/well)in 1 mM EGTA pH 7.4 is adsorbed onto PVC 96-well microtiter plates for 1hr. The plates are then washed 3 times with, and all wells exposed for10 min to, 0.5% BSA in TBS (10 mM Tris, 140 mM NaCl and 0.02% NaN₃, pH6.9) in order to block non-specific protein binding. The second layer,100 μl/well of the blood samples diluted (1:500) into BSA-TBS, is addedand the sample is incubated for 2 hr. Serving as quantitation standards,duplicate 100 μl/well samples of human IgG or 2E2 diluted over a rangeof concentrations from 0.003-3.0 μg/ml are also similarly plated andincubated. The plates are washed with a Solution A, containing 0.5% BSA,10 mM sodium phosphate, 145 mM NaCl, 1.5 mM MgCl₂, 0.05% triton X-100and 0.02% NaN₃, pH 7.2. Then 50 μl/well of affinity-purifiedbiotinylated goat anti-human IgG diluted 1:500 in Solution A is addedand incubated for 1 hr. After washing, 50 μl/well ofstreptavidin-alkaline phosphatase, diluted (1:200) in Solution A, isadded, incubated for 1 hr and removed. Then 50 μl/well of thecolorimetric reaction mixture, comprised of the substratepara-nitrophenylphosphate (1 mg/ml) in substrate buffer (50 mM Na₂CO₃,50 mM NaHCO₃ 1 mM MgCl₂ at pH 9.8), is added. After 6-8 min the reactionis stopped with 1M sodium hydroxide (50 μl/well). All steps areperformed at room temperature. The reaction endpoint is measured with anELISA reader at a wavelength of 405 nm. Each determination is done induplicate.

Antibodies:

The hybridoma cell line secreting mAb 2E2 is generated using standardhybridoma technology by fusing spleenocyctes obtained from a transgenicmouse, strain HCo7/Ko5, following its immunization with benzoylecgonine(BE) coupled to 1,4-butanediamine-derivatized keyhole limpet hemocyanin(KLH) with the mouse cell line P3X63-Ag8.653. Production of mAb 2E2 isaccomplished by growing hybridomas in severe combined immunodeficient(SCID) mice and collecting the ascites fluid. The hybridoma-secreted mAbis purified from ascites by sodium sulfate precipitation and a severalstep protein A-Sepharose column chromatography procedure adapted fromthat previously described. Identification of the full length amino acidsequences of the polyacrylamide gel separated heavy and light chains ofthe 2E2 molecule is accomplished using liquid chromatography/massspectroscopy (LC/MS/MS) analysis of their tryptic fragments. The heavy(H) chain is identified as a γ₁ protein of the human VH3 family geneDP-50. The light (L) chain is identified as a mouse λ VL2. The MSsequencing is consistent with and confirmed results obtained previouslyfrom Edman degradation NH₂-terminal sequencing of the Western blotted Hand L chains as well as the sequencing of mRNA-dependent cDNArepresenting the 2E2, V_(H) and V_(L) chain regions. The γ1 human Hchain NH₂-terminal sequence is: EVQLVESGGGLVKPGGSLRL- (see SEQ ID NO:2), while the mouse λ chain NH₂-terminal sequence is:QAVVT/IQESALTTSPGGTV- (see SEQ ID NO: 1). Although the 2E2 hybridomacontains the recombined DNA sequence for a human κ L6 light chain, andthis is consistent with the human κ chains of anti-digoxin antibodiesgenerated from these transgenic mice in previous work, the L chain forthe mAb expressed and used is a murine λ. These results are consistentwith a recent report that hybridomas from the HCo7/Ko5 strain oftransgenic mouse can generate mixed-chain, human H, mouse L mAbs inaddition to human sequence mAbs. Overall, 2E2 has about an 87% sequenceidentity/homology with human IgG(λ)1 immunoglobulins.

The murine anti-cocaine mAb 3P1A6 obtained from BioDesign International,Inc., (Saco, Me.) has previously been reported to have a high affinity(K_(d)=0.2 nM) for cocaine and approximately 12-fold and 1.500-foldlower affinities for the inactive metabolites benzoylecgonine (BE) andecgoninemethylester (EME), respectively. The murine anti-cocaine mAbB4E10 has been determined to have a moderate affinity for cocaine(K_(d)=40 nM) and approximately 30-fold and 50,000-fold lower affinitiesfor BE and EME, respectively. Therefore, the murine mAbs and 2E2 havesimilar specificities for cocaine over its major metabolites, but anapproximately 200-fold range difference for cocaine affinity. As anadditional control, to test for non-specific in vivo effects resultingfrom infusion of mAb, non-specific human polyclonal IgG immunoglobulinis administered to mice. These latter immunoglobulins have no measurableaffinity for cocaine or its major metabolites (data not shown).

Solid Phase Extraction of Cocaine and Metabolites from Plasma and Brain.

In order to determine in vivo concentrations of cocaine and itsmetabolites BE and EME, following the i.v. injection of cocaine, 100-400μl samples of heparinized/NaF treated plasma and 400 μl samples of brainhomogenates obtained from cocaine-treated animals are added to 2 ml of0.1 M Na phosphate buffer, pH 6.0. This is followed by the addition of5% trichloroacetic acid at a volume equal to that of the experimentalsample (100-400 μl). These mixtures are shaken for 20 minutes and thencentrifuged for 15 min at 7000 rpm, all at room temperature, in order toprecipitate the denatured protein. The supernatants are collected andadjusted to pH 5.4 with 10 M NaOH. Then to serve as internal standardsfor establishing the identification of cocaine and its metabolites aswell as for normalization of the recovery of cocaine/metabolites fromthe mouse samples, 50 μl of a sample containing deuterated (D₃)cocaine-D₃, BE-D₃ and EME-D₃ (each at 1 μg/ml) is added to all of theexperimental and the standard control samples before their undergoingsolid-phase extraction/column elution. Duplicate, standard control tubes(2 ml) are also prepared containing; 0.1 M Na phosphate buffer, 50 μl ofthe internal standards D₃ cocaine/BE/EME (1 μg/ml, each), 200 μl ofnormal mouse plasma and varying amounts of cocaine (1-500 ng) and usedto generate the standard cocaine concentration curves. Similarly,standard concentration curves are also generated for BE and EME. Also 10μl of the stock solution of cocaine HCl (0.139 mg/ml) that is infusedinto the mice is also mixed with the phosphate buffer and thecocaine-D₃/metabolite-D₃ standards for quantification of the cocaineadministered to the animals. Thus, the cocaine/metabolite levels aredetermined relative to that of standard samples undergoing the samecolumn extraction, elution and derivatization procedures.

The procedure of Varian is used to extract and recovercocaine/metabolites from the plasma and brain samples and standards.First, Bond Elut Certify columns with the non-polar C8 sorbent, set in aVarian vacuum manifold are conditioned by washing with 2 ml methanol,followed by 2 ml of 0.1 M Na phosphate buffer, pH 6.0. Next, theprepared plasma and brain homogenate samples (2 ml) are loaded onto theBond Elut columns. The columns are then washed with 6 ml of deionizedwater, 3 ml of 0.1 M HCl, and 9 ml of methanol. The column-boundanalytes are then eluted with 2-3 ml of a freshly prepared solution ofdichloromethane:2-propanol:ammonium hydroxide (mixed: 78:20:2, v/v/v).These extracts are then evaporated to dryness under nitrogen at 45° C.for 15 min. The residue samples are derivatized with 25 μlN-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) mixed with 25 μlethyl acetate. These samples are vortexed and incubated at 65° C. for 30min. After cooling, the trimethylsilyl-derivatized samples aretransferred to glass autosampler vials for analysis by GC/MS. The GC/MSanalysis of analytes is typically completed within 1-2 hours of samplederivatization. Analyses carried out more than eight hours afterderivatization are discarded.

Gas Chromatography/Mass Spectrometry.

The gas chromatograph/mass spectrometer (GC/MS) consisted of a ShimadzuGC 17A series GC, interfaced with a Shimadzu QP-5050A quadruple MS fixedin an electron impact ionization mode with selective ion monitoring. TheGC/MS is operated with a transfer line temperature of 280° C. and asource temperature of 280° C. The MS is calibrated on a daily basisusing perfluorotributylamine. The electron multiplier voltage is set at1.2 kV. Chromatographic separation is achieved using a Restek Rtx-5MScross linked 5% diphenyl-, 95% dimethylsiloxane capillary column (30m×0.25 mm i.d, 0.25 μm film thickness). Helium is the carrier gas andused at a flow rate of 1.0 ml/min.

A Shimadzu AOCs autosampler is used to inject 2 μl of extract sampleinto the GC/MS. The GC, equipped with split/splitless injection port, isoperated at 280° C. in the splitless mode with a high pressure injectionset at 150 kPa for 0.75 min. The oven temperature profile is establishedas follows: the initial temperature is 100° C. and it is held for 1 min,then increased at a rate of 20° C./min up to 320° C. This temperature isheld for 8 min resulting in a total run time of 20 min. The lower limitsof cocaine/BE/EME detection ranged from 1-5 ng/ml and the linear dynamicrange for most analytes is 1-3000 ng/ml. The instrument performance isevaluated by analysis of the calibrator and control samples. Analytesare identified and their concentrations are determined using both theinternal deuterated standards and concentration control samples preparedwith normal mouse serum, respectively, as described above. The responsefactor is determined for each analyte. The response factor is calculatedby dividing the area of the analyte peak by the area of the internalstandard peak. Calibration curves are then prepared by plotting a linearregression of the analyte/internal standard response factor versus theanalyte concentration for all calibrators analyzed. The standard curveis constructed using a set of cocaine/metabolite samples varying over aconcentration range of 1-500 ng/ml. The standard curve is used todetermine concentrations of analytes in both control and experimentalsamples.

Chemicals Reagents and Reference Standards:

Standard solutions of cocaine, BE and EME (each 1 mg/ml) are prepared inmethanol or acetonitrile and serve as stock solutions for preparing thereference standard curves. The cocaine-D₃, BE-D₃ and EME-D₃ are used asthe internal standards (0.1 mg/ml each in methanol or acetonitrile).MSTFA is the derivatizing reagent. Normal mouse plasma with heparin isobtained. The human hemoglobin standards and control standards areobtained. All other chemicals and immunoreagents are purchased. Allreagents and organic solvents are of analytical grade or HPLC grade.

Data Analysis and Statistics:

Cocaine and 2E2 pharmacokinetic data are analyzed using the programWinNonLin. The program provides Akaike Information Criterion (AIC) andSchwartz Bayesian Criterion (SBC) measures of “goodness of fit” of thedata to the one or two compartment pharmacokinetic models that are used.Data are first analyzed according to a single compartmentpharmacokinetic model. In some experiments a single compartment modelgave a poor fit to the cocaine pharmacokinetic data, as assessed by asystematic deviation of the model from the data. In these cases the fitto the data is improved by applying a two compartment pharmacokineticmodel that assumes cocaine distributed between a central and aperipheral compartment. In addition to an improvement in the AIC and SBCmeasures, the improvement of the fit of the model to the data isevaluated by a lack of a systematic deviation from the data points and aconcomitant reduction in the sum of squares residuals. Applyingpharmacokinetic models that assumed that cocaine distributed betweenmore than two compartments only slightly improved the fit to theobserved data and this additional complexity is considered unnecessary.Statistical comparisons of the cocaine and metabolite levels observed inthe presence and absence (vehicle) of antibody at the single 5 min timepoint used non-parametric Mann-Whitney test while the Analysis ofVariance (ANOVA) procedure is used to compare the results obtained overdifferent experimental days.

Results

The Pharmacokinetics of mAb 2E2.

In determining the pharmacokinetics of mAb 2E2 in mice, the firstsamples of tail vein blood are taken 3 minutes after completion of thei.v. infusion of 2E2 (120 mg/kg) via the jugular vein of mice. Theinitial mean±SEM blood concentration of mAb is determined to be 370±17μg/ml (n=8 mice). As shown in FIG. 1 there is no evidence for an initialdecrease in blood concentrations over the first 24 hours. Indeed, 2E2concentrations increased slightly over the first four hours and thenappeared to plateau for approximately 20 hours. The mean concentrationof 2E2 as measured 24 hours after infusion is 422±21 μg/ml. After 24hours, the concentrations of 2E2 in blood then begin to decline and thisis adequately described by a single compartment pharmacokinetic modelwith an elimination t_(1/2) of 8.1 days (FIG. 1). This model gives acalculated volume of distribution at steady state (Vdss) for 2E2 in thisgroup of mice of 0.28 l/kg.

The Plasma Pharmacokinetics of Cocaine.

Next, the disposition of cocaine in mouse plasma subsequent to its i.v.injection via the jugular vein is determined. The highest plasmaconcentrations measured (˜110 ng/ml) are observed at the earliest sampletime, after which cocaine concentrations declined rapidly (FIG. 4A). Apharmacokinetic model assumes that cocaine distributed between a centraland a peripheral compartment improves the fit to the observed data ascompared to a single compartment model. This result is similar to thatwhich has previously been reported for i.v. injected cocaine in severalspecies including rats, non-human primates and for i.p. injected cocainein mice. The simplest pharmacokinetic model that provides a generaldescription of the data generated parameter estimates for thedistribution half-life (t_(1/2α)) and terminal elimination half-life(t_(1/2β)) for cocaine of 1.9 and 26.1 min, respectively. The calculatedVdss is 6.0 l/kg.

Effect of Cocaine-Specific mAbs on Cocaine Distribution.

In these experiments a single time point, 5 minutes after i.v. cocaineadministration, is selected at which to determine the effect ofcirculating anti-cocaine mAbs on the in vivo plasma and brain levels ofcocaine. As shown in Table 1 below, at 5 minutes considerabledistribution of cocaine has occurred as the plasma concentration (˜77ng/ml) declines from an initial value of ˜110 ng/ml (45 sec, FIG. 4A)and brain levels (˜1070 ng/g) are about 10-fold higher than that inplasma. The presence of mAb 2E2 then produced a substantial 29-foldincrease in plasma and an almost 5-fold decrease in brain cocaineconcentrations (Table 1) in comparison to the vehicle controls.Furthermore, pretreatment with the mouse anti-cocaine mAbs 3P1A6(K_(d)=0.2 nM) and B4E10 (K_(d)=40 nM,) also similarly increased cocaineconcentrations in plasma, while they are somewhat less effective than2E2 in decreasing cocaine concentrations in the brain. These resultsclearly demonstrate the capability of cocaine-specific mAbs for in vivobinding of cocaine. In contrast, the pretreatment with non-specifichuman polyclonal antibodies with no measurable affinity for cocaineproduced a small increase in cocaine concentrations in both plasma andbrain relative to those in mice pretreated with the vehicle (PBS).

TABLE 1 Change Change Plasma cocaine from Brain cocaine fromconcentration (ng/ml) vehicle concentration (ng/g) vehicle Vehicle 76.6± 3.3 (n = 23)  1070.5 ± 32.1 (n = 22)   Human IgG 121.2 ± 5.6* (n =6)    +1.6-fold  1568 ± 130.5* (n = 5) +1.5-fold 2E2 2197.7 ± 75.8** (n= 6)  +28.7-fold 223.7 ± 25.5** (n = 6) −4.8-fold 3P1A6 2215.5 ± 157.2**(n = 6) +28.9-fold 469.2 ± 68.9** (n = 6) −2.3-fold B4E10 1591.5 ±57.8** (n = 6)  +20.8-fold 560.5 ± 62.4** (n = 6) −1.9-fold

The Dose-Dependent Effect of 2E2 on Plasma and Brain CocaineConcentrations.

In view of the magnitude of the effects of stoichiometric doses of theanti-cocaine mAbs on the plasma and brain cocaine concentrations, thedose-dependency for the responses is determined using mAb 2E2. In theabsence of 2E2 the mean±SEM plasma cocaine concentration at 5 min postcocaine injection is 78.5±4.5 ng/ml (FIG. 2A). Infused 2E2 produces adose-dependent increase in plasma cocaine concentrations (FIG. 2A). Thelowest dose of 2E2 (12 mg/kg, a 1:10 mAb:cocaine ratio) produces asignificant (p<0.01, one-way ANOVA with post-hoc test) 5.1-fold increasein plasma cocaine concentrations while the highest dose (360 mg/kg, a3:1 ratio) produces a dramatic 46.1-fold increase in cocaineconcentrations. The calculated dose of 2E2 that produces 50% of thehighest effect for the range of 2E2 doses used (ED₅₀) is approximately80 mg/kg, a somewhat less than stoichiometric amount of 2E2.

In the absence of 2E2 the mean±SEM brain cocaine concentration at 5 minpost injection, corrected for cocaine present in cerebral blood, is796.8±50 ng/ml (FIG. 2B). This represents a brain:plasma cocaineconcentration ratio of 10:1. 2E2 then produces a dose-dependent decreasein brain cocaine concentrations (FIG. 2B). At the dose of 24 mg/kg, 2E2produces a significant 35% decrease in cocaine concentrations. At the2E2 dose of 360 mg/kg, after correction for cocaine present in theresidual blood, the brain cocaine concentration is negligible. The ED₅₀for the range of 2E2 doses used is approximately 60 mg/kg.

The Effect of 2E2 on Cocaine Metabolite Concentrations in Plasma andBrain.

An additional point of interest is the determination of the effects ofcirculating 2E2 on the in vivo metabolism of cocaine. As shown in FIG.3, at 5 min after the injection of cocaine in the absence of 2E2, meanconcentrations of the predominant cocaine metabolite in mice, EME, whichresults largely from plasma butyrylcholinesterase activity, are 25 ng/mland 267 ng/g in plasma and brain, respectively. This represented abrain:plasma ratio for EME of 10.7:1, a ratio similar to that of cocainein these mice (FIG. 2), thus the cocaine:EME ratio is ˜3:1 in bothplasma and brain. Produced by non-specific liver carboxylesteraseactivity, BE levels are lower with mean concentrations of 7 ng/ml and 16ng/g in plasma and brain, respectively, representing a brain:plasmaratio of 2.3:1. A modest increase (˜3-fold) in plasma BE concentrationsis observed with increasing doses of 2E2 but the effect approached aplateau at 2E2 doses above 40 mg/kg. There is a concomitant decrease inbrain BE concentrations which is observed at doses above 40 mg/kg. Theseresults are consistent with mAb 2E2 having no effect on BE productionbut a sufficiently high affinity for BE to sequester some in the plasma,but its levels are limited. In contrast, plasma EME concentrationsappear unaffected at the lower doses of 2E2, but an approximate 2-foldreduction is observed at the highest dose of 2E2. There is no systematiceffect of 2E2 dose on brain EME concentrations (FIG. 3). It is of note,that despite 2E2's effective in vivo binding of cocaine, its alterationsin cocaine's initial metabolism appear modest.

Effect of 2E2 on the Pharmacokinetics of Cocaine in Plasma and Brain.

Next, the effects of a stoichiometric dose of 2E2 on thepharmacokinetics of a single injection of cocaine are determined. Asshown in FIG. 4A, in the presence of 2E2, the peak plasma concentrations(˜1,100 ng/ml) of cocaine are observed at the earliest time point (45sec) sampled after its injection. This is similar to what is observed inthe absence of 2E2. However, the peak plasma concentration is 11.3-foldhigher in the presence than in the absence of 2E2. Furthermore, incontrast to the biexponential decrease in the concentrations of cocaineobserved in the absence of 2E2, the decrease in the cocaineconcentration is well described by a pharmacokinetic model that assumesa single compartment and no initial distribution phase. Thus, thecalculated t_(1/2) for the disappearance of cocaine from plasma in thepresence of 2E2 is 17.1 min and this contrasts with the distribution andelimination phases, with parameter estimates for t_(1/2α) and t_(1/2β)of 1.9 and 26 min, respectively. 2E2 also produces a sustained increasein the plasma cocaine concentration that results in 26-fold increase inthe area under the concentration-time curve (AUC) in plasma. Consistentwith this result, the calculated Vdss of cocaine in the presence of 2E2is 0.2 l/kg as compared to 6.0 l/kg in the absence of 2E2.

As shown in FIG. 4B, the cocaine concentration-time profile in braindiffers substantially from that observed in the plasma (FIG. 3A). Theconcentration of cocaine in the brain (corrected for cocaine present inresidual blood) at 45 sec (˜650 ng/g) after the injection isapproximately 6-fold higher than that measured in plasma. The braincocaine concentrations subsequently increase further and the highestmeasured concentration is observed at 3 min (˜1,500 ng/g), after whichconcentrations then rapidly decline. A pharmacokinetic model thatassumes a first-order input to the brain and a first-order output isused to describe the increase and subsequent decrease in brain cocaineconcentrations. The estimated t_(1/2) for entry into the brain isapproximately 2.0 min. Furthermore, a pharmacokinetic model assuming twocompartments described the disappearance of cocaine from the brain. Theparameter estimates for t_(1/2α) and t_(1/2β) are 2.0 min and 14.5 min,respectively (FIG. 4B), values similar to those obtained for the plasma.

In the presence of 2E2, the peak cocaine concentration (˜490 ng/g) isobserved at the earliest sample time and it subsequently declinesrapidly over time (FIG. 4B). There is no indication of the normaldelayed influx and peak of the cocaine concentrations in the brain and asingle compartment model approximates the decline in cocaineconcentrations. The estimated t_(1/2) is 3.8 min, a value considerablyfaster than the t_(1/2β) value obtained in the absence of 2E2.Importantly, 2E2 produces an approximately 4.5-fold (78%) decrease inthe cocaine AUC in the brain.

Discussion

The low volume of distribution of 2E2 observed in mice is similar tothat previously reported for several murine and rat monoclonal IgG₁antibodies and human polyclonal IgG₁ antibodies in rats and isconsistent with 2E2's distribution being predominantly restricted to theblood volume. Additionally, the elimination t_(1/2) value for 2E2 isrelatively long and similar to that reported for other murine, rat andhuman antibodies in rats. This indicates that 2E2's effects on cocainepharmacokinetics could persist for several days after a singleinjection. Furthermore, the terminal elimination t_(1/2) of cocaine ismore than 400-fold faster than that of 2E2 and, therefore, it can beassumed that the plasma concentration of 2E2 is constant during thestudy of cocaine pharmacokinetics. Interestingly, although the V_(d) andt_(1/2) for 2E2 are similar to those previously described for antibodiesin rodents, there is no evidence for an initial distribution of 2E2 fromthe blood to the interstitial spaces.

As to 2E2's in vivo binding of cocaine, its effect on the plasmaconcentration of cocaine is saturable, which is consistent with thelimited number of cocaine molecules present. Furthermore, doses of 2E2that are only 10% to 30% of the dose of cocaine still provide ameasurable increase in plasma cocaine concentrations and a decrease inexposure of the brain to cocaine. This is consistent with reports that a0.3 molar ratio of anti-phencyclidine (PCP) Fabs decreases thebehavioral effects of PCP in rats. Furthermore, a 4 mg dose of ananti-cocaine mAb, representing a molar ratio of approximately 0.005, hasbeen reported to antagonize the behavioral effects of repeated 1 mg/kgdoses of cocaine HCl. While, 30 and 40 mg/kg doses of another murineanti-cocaine mAb decrease the self-administration of cocaine at molarratios of approximately 0.2 for each cocaine injection. The finding that2E2 produces a substantial reduction in the brain's exposure to cocaineat equimolar ratios and has measurable effectiveness at lower molarratios indicates that 2E2 will reduce brain concentrations even after amAb dose has been partially eliminated. Thus the efficacy of a givendose of 2E2 is prolonged.

The demonstration that an equimolar dose of a nonspecific antibody didnot significantly alter either plasma or brain concentrations of cocainerules out the possibility of nonspecific effects of infused IgG proteinsas an explanation for the mAb effects on cocaine pharmacokinetics.Therefore, the efficacy of anti-cocaine mAbs requires specificity of thebinding interaction between the drug/antibody molecules. However, thethree anti-cocaine mAbs with affinities ranging from very high(K_(d)=0.2 nM) to modest (K_(d)=40 nM, as measured in vitro atequilibrium) are approximately equipotent under the limited in vivoexperimental conditions tested. This suggests that the ability of anantibody to influence the pharmacokinetics of cocaine may not be highlyaffinity sensitive. Therefore, antibodies with a fairly broad range ofaffinities may have clinical efficacy. Antibodies with low affinity,that is having K_(d)s in the μM range, have been reported to amelioratesome behavioral effects of cocaine in rodents, but would most likely notbe as effective as 2E2 on treating cocaine-related disorders.

In the presence of 2E2, the initially observed approximately 10-foldincrease in the concentration of cocaine in plasma, the lack of aninitial distribution phase from the plasma and the reduction of the Vdssof cocaine to essentially that of 2E2 are all consistent with 2E2restricting cocaine's distribution predominantly to the blood volume.Therefore, the 2E2-induced decrease in brain cocaine concentrations isdue to an inhibition of cocaine distribution from the blood to thebrain. Furthermore, the reduction in the peak levels and thedistribution of cocaine to the brain occurs at all time points,indicating that 2E2 did not simply delay cocaine's distribution to thebrain. This report is the first to demonstrate that an anti-cocaine mAbcan prevent the entry of cocaine into the brain and it is consistentwith previous reports that active immunization-induced anti-cocaineantibodies decrease cocaine levels in brain after i.v., intranasal ori.p. cocaine administration. The ability of 2E2 to decrease brainconcentrations of cocaine is also consistent with the mAb-inducedreductions observed for other psychoactive drugs such as phencyclidine,methamphetamine and nicotine in rats.

The markedly altered distribution of cocaine is the result of cocainebinding to 2E2. While it may initially be believed that this mAb bindingof cocaine also restricts cocaine's access to the enzymes thatmetabolize it, thereby decreasing its clearance, there is no evidence ofan increase in the elimination t_(1/2) of cocaine in plasma. This isconsistent with the reported lack of effect of active immunization oneither the elimination of cocaine from plasma or on the rate ofmetabolism of nicotine. However, a murine anti-nicotine mAb and activeimmunization against nicotine have also been reported to significantlyincrease the elimination t_(1/2) of nicotine in rats. The reasons forthese discrepant results are not clear at present but do not appear tobe related to different affinities of the mAbs or the polyclonalantibodies for their target molecules. If anti-drug antibodies caninhibit the metabolism and slow the rate of drug elimination this wouldincrease the in vivo concentrations resulting from repeated drug dosesand may not be desirable for an immunotherapeutic agent. Importantly,the lack of effect of 2E2 on cocaine elimination from plasma shouldminimize the potential for 2E2 to become saturated following repeateddoses of cocaine.

In summary, the high affinity anti-cocaine mAb 2E2 limits thedistribution of cocaine to the plasma thus decreasing the levels ofcocaine reaching the brain without any detectable effect on the rate ofelimination of cocaine. The data further supports the general concept ofthe usefulness of immunotherapy for the treatment of drug abuse and isconsistent with mAb 2E2 being effective as a passive immunotherapy forthe prevention of relapse in cocaine abuse.

Example 2 2E2 Heavy Chain Gene Cloning into Expression Retrovector

2E2 heavy chain CDS was amplified from plasmid in a single PCR reactionusing primers 2E2HCl (SEQ ID NO: 15) and INHC2 (SEQ ID NO: 16). Primer2E2HCl added a Hind III site at the 5′ end and Kozak translationinitiation sequence just before the translation start codon of thesignal peptide. Primer INHC2 encoded silent mutations in the 3′ end ofthe heavy chain gene to remove a potential mRNA splicing site andcontributed an Xho I site for cloning. The obtained PCR product wasdigested with Hind III and Xho I restriction nucleases and ligated intothe retrovector plasmid pCS-newMCS-WPRE (new ori) which had also beendigested with the same two enzymes. Clones of the resulting constructwere DNA sequenced through the assembled heavy chain gene and theflanking regions and clone 15 was confirmed to encode valid 2E2 heavychain CDS.

2E2 Light Chain Gene Cloning into Expression Retrovector.

2E2 light variable region CDS was amplified from plasmid using primers2E2LC1 (SEQ ID NO: 17) and 2E2LC3 (SEQ ID NO: 18). Primer 2E2LC1 added aHind III site at the 5′ end and Kozak translation initiation sequencejust before the translation start codon of the signal peptide. Primer2E2LC3 set up the amplified variable region sequences for in-framefusion to the CPS-M human lambda 2 light chain constant region CDS(hIGLC2). In the second PCR reaction, hIGLC2 light constant region wasamplified and set up for fusion with 2E2 light variable region usingprimers 2E2LC2 (SEQ ID NO: 19) and 2E2LC4 (SEQ ID NO: 20) andNewSP7Fixed-MCS-Lambda2 (CPS-M, Somerset, N.J.) plasmid as a DNAtemplate. Primer 2E2LC2 was a reverse compliment to primer 2E2LC3 andhence served the same purpose of setting up the amplified 2E2 lightchain sequences for fusion. Primer 2E2LC4 encoded the 3′ end of thelambda-2 light chain constant region and contributed an Xho I site forcloning. The amplified products from PCR reaction 1 and 2 were used as aDNA template with the outermost primers 2E2LC1 and 2E2LC4 to amplifyfull-length 2E2 light chain CDS. The obtained PCR product was digestedwith Hind III and Xho I restriction nucleases and ligated into theretrovector plasmid pCS-newMCS-WPRE (new ori) (CPS-M, CPS-M, Somerset,N.J.) which had also been digested with the same enzymes. Clones of theresulting construct were DNA sequenced through the assembled light chaingene and flanking regions. Clone 3 was confirmed to encode validfull-length 2E2 light chain CDS.

Cell Line Creation

Retrovector Production.

Cells from the 293GP working cell bank (Pangenix, San Diego, Calif.)were cultivated in DF medium. 293GP cells were passaged to 8 T150 flasksusing trypsin (HyClone Catalog #SH30042). Two hours prior totransfection, the flasks were changed to 12.5 mL of DF medium.Transfection was performed using 864 μg of the 2E2 antibody light chain(pCS-2E2ChimericLLC-WPRE (new ori)) and 54 μg of expression plasmid forVesicular Stomatitis Virus envelope glycoprotein or 1728 μg of the 2E2antibody heavy chain (pCS-2E2HC-WPRE (new ori)) and 108 μg of expressionplasmid for Vesicular Stomatitis Virus envelope glycoprotein. Theplasmid solutions were combined with cell culture grade water for atotal volume of 17.47 mL and 2.52 mL of 2M CaCl₂ and then precipitatedby dropwise addition into 19.92 mL of 2×HBS solution. 2.5 mL ofsuspension was added to each of the 8 T150 flasks and incubated on thecells for six hours at 37° C. in a 5% CO₂ atmosphere. Growth at 37° C.±1° C. in a 5%±1% CO₂ atmosphere will be referred to as standardconditions from this point forward. The final LC and HC transductionessentially followed the above process, except 16 T150 flasks were usedinstead of only 8 T150 flasks.

After six hours, the culture medium was replaced with 20 mL of fresh DFmedium. The flasks were incubated under standard conditions until thesecond day after transfection. The medium was collected from the 8 or 16T150 flasks and filtered through a 0.2 micron filter. The retrovectorwas concentrated from the 160 mL or 320 mL of harvested medium bycentrifugation in a Beckman J-301 centrifuge with a JA-30.5 rotor at18,750 rpm (40,000×G) for 90 minutes at 4° C. The supernatant wasaspirated from the centrifuge tubes and the pelleted material in eachtube was resuspended in 25 μL of PF CHO LS medium. The concentratedvector was diluted 1:5 with PF CHO LS medium. The 1:5 diluted vector andthe concentrated vector was used for the GCHO transduction step.

Transduction of GCHO Cells with Retrovector.

Parental GCHO cells were established in culture and prepared fortransduction. Two tubes containing a suspension of 6×10⁴ viable GCHOcells were prepared in 5 mL of PF CHO LS medium with 8 μg/mL ofpolybrene. This cell suspension was incubated under standard conditionsfor a minimum of two hours prior to the addition of the retrovector.Immediately prior to the addition of retrovector, the cell suspensionwas centrifuged for four minutes at 1500 rpm (500×G) in a tabletopcentrifuge (Beckman Coulter Allegra 6 with a GH 3.8 rotor) and thesupernatant was removed without disturbing the cell pellet. Theretrovector was added to the GCHO cell pellet, mixed and then incubatedunder standard conditions.

After one day of incubation, 5 mL of phosphate buffered saline was addedto the tube containing the cell-retrovector mixture. This mixture wasthen centrifuged for four minutes at 1500 rpm (500×G) in a tabletopcentrifuge. The supernatant containing any residual retrovector wasremoved and another wash and spin were performed. The supernatant wasagain removed without disturbing the cell pellet, and the cells wereresuspended in 2 mL PF CHO LS medium. The suspended cells were seededinto a 12-well cell culture plate and expanded over the following daysthrough consecutive passages into successively larger cell cultureflasks using PF CHO LS medium.

Each subsequent transduction was performed using cells in culture fromthe previous transduction. The transductions were performed followingthe same methods mentioned above. The pooled population from eachtransduction was expanded and cryopreserved. A protein sample was takenfor analysis by ELISA, and a sample of cells was submitted for gene copyindex and retrovector component testing.

Fed Batch Production of h2E2 Antibody from the Pooled Population ofCells.

Cell line GCHO/sC-2E2 Ab-LC4/HC5-R was scaled up for production by fedbatch analyses in shake flasks. Duplicate 250 mL shake flasks wereseeded with 300,000 viable cells per mL in 60 mL working volume of PFCHO LS media. The cultures were terminated when the viability fell belowfifty percent.

Establishment of Clonal Cell Lines.

Clonal selection was performed on an aliquot of GCHO/sC-2E2 Ab-LC4/HC5-Rcells. The cells were diluted to 0.5 and 0.75 viable cells per 200 μL inPF CHO LS medium with 2% FBS. Thirty 96-well plates were seeded with 200μL per well of cell suspension for each of the dilutions (SOPSTM-CEL-0330). The seeded 96-well plates were incubated under standardconditions and were observed microscopically on two different days forthe development of approximately 400 colonies originating from singlecells (SOP STM-CEL-0330). Media was collected on Day 14 from wells inwhich single colonies were observed. Media was replaced with PF CHO LS.The media samples were screened by ELISA for protein titer.

Selection and Testing of the Clonal Cell Lines.

The top 20 clones were selected based on antibody titer. The selectedclonal cell lines were tested for productivity in a fed batch overgrowthstudy. Duplicate 250 mL shake flasks were seeded with 300,000 viablecells per mL in 60 mL working volume of PF CHO LS media. The cultureswere terminated when the viability dropped below fifty percent.

Twenty-five vials of each clonal cell line were prepared forcryopreservation. Five vials of each clone were used for QC testing. TheQC tests included viability, gene copy index, retrovector component,bioburden, and mycoplasma.

Production.

Clonal cell lines were made by performing limited dilution cloning ofthe GCHO/sC-2E2 Ab-LC2/HC3-R pooled population. Twenty clonal cell lineswere screened by fed batch overgrowth analysis. Protein titers weredetermined by Protein A HPLC analysis. The average maximum protein levelof the 20 clones was 562 mg/L compared to 466 mg/L for the pool. The topclones, #85, 188, 258, 275 and 323 were primarily selected based ontiter. These top clonal lines had maximum expression levels ranging from697 mg/L for clone #275 to 833 mg/L for clone #85 and averaged 753 mg/Lcompared to 466 mg/L for the pool. The mean maximum viable cell densityof the 20 clones was 68.2×10₅ viable cells/mL. The top clones reachedmaximum cell densities on days 4 and 6 with peak viable cell densitiesranging from 43.6×10⁵ for clone #275 to 130.2×10⁵ viable cells/mL forclone #188.

Example 3 Comparison of 2E2, h2E2 (Human Kappa Light Chain ConstantRegion) and h2E2 (Human Lambda Light Chain Constant Region)

Production levels were compared for the original 2E2 murine hybridoma,recombinant 2E2, h2E2 (human kappa light chain constant region) and h2E2(human lambda light chain constant region). Results are shown in Table2, below.

The original murine hybridoma cell line that produced mAb 2E2 when grownin culture produced mAb at a level of approximately 0.7 μg/ml versus anexpected 10-20 μg/ml for standard murine mAb producing hybridoma.Large-batch, or scale-up culturing of the hybridoma cell line provedunsuccessful. The required growth or culture media included fetal calfserum which meant purification of 2E2 was difficult and contaminatingbovine serum proteins and antibodies precluded use of the mAb forclinical production.

The murine hybridoma 2E2 was also grown in vivo in SCID mice and mAb wascollected in ascites fluid with production levels ranging from 100-150μg/ml of mAb versus the 1,000-2,000 μg/ml generally obtained forstandard murine mAbs. Further, the mAb needed to be purified from theascites fluid and removal of murine serum proteins and murine antibodieswas problematic.

The production of a recombinant form of 2E2 (murine lambda light chainconstant region) and a first version of humanized 2E2 (human kappa lightchain constant region) was achieved in transiently transfected COS-7 andHEK293T cells. However, the levels of production were 0.2-0.5 μg/ml. Thecells could be grown in defined serum free media, but production levelswere at least 10-fold lower than that of a control mAb produced with thesame technology. Stably transfected cells expressing mAb as needed forlarge-scale production were not achieved.

Surprisingly, 20 stably transfected GPEx® CHO parental cell lines wereisolated that produced mAb h2E2 (human lambda light chain constantregion) at levels that averaged 562 μg/ml. The top five cell linesproduced h2E2 at levels 697-833 μg/ml. The cells are grown in serum freedefined media and mAb is isolated to a high level of purity usingstandard Protein-A column chromatography.

TABLE 2 Comparison of Production Levels for 2E2 and h2E2 mAbs ConstantmAb region Production Level 2E2 (murine hybridoma) Murine lambda 0.7μg/ml grown in culture 100-150 μg/ml grown in vivo 2E2 (recombinant)Murine lambda 0.2-0.5 μg/ml h2E2 (recombinant) Human kappa 0.2-0.5 μg/mlH2E2 (recombinant) Human lambda 562 μg/ml

The foregoing description of various embodiments and principles of theinvention has been presented for the purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinventions to the precise forms disclosed. Many alternatives,modifications, and variations will be apparent to those skilled the art.Moreover, although multiple inventive aspects and principles have beenpresented, these need not be utilized in combination, and variouscombinations of inventive aspects and principles are possible in lightof the various embodiments provided above. Accordingly, the abovedescription is intended to embrace all possible alternatives,modifications, aspects, combinations, principles, and variations thathave been discussed or suggested herein, as well as all others that fallwithin the principles, spirit and scope of the inventions as defined bythe claims.

What is claimed is:
 1. A monoclonal antibody that specifically bindscocaine, wherein said antibody comprises: (a) a murine lambda lightchain variable region CDR1 comprising SEQ ID NO: 4; (b) a murine lambdalight chain variable region CDR2 comprising SEQ ID NO: 5; (c) a murinelambda light chain variable region CDR3 comprising SEQ ID NO: 6; (d) ahuman gamma heavy chain variable region CDR1 comprising SEQ ID NO: 7;(e) a human gamma heavy chain variable region CDR2 comprising SEQ ID NO:8; (f) a human gamma heavy chain variable region CDR3 comprising SEQ IDNO: 9; and (g) a human light chain constant region.
 2. The monoclonalantibody of claim 1, wherein the human light chain constant region isselected from the group consisting of a human kappa light chain constantregion and a human lambda light chain constant region.
 3. The monoclonalantibody of claim 2, wherein the human light chain constant region is ahuman lambda light chain constant region comprising SEQ ID NO:
 22. 4.The monoclonal antibody of claim 1, wherein the antibody has a murinelambda light chain variable region comprising SEQ ID NO: 1 and a humangamma heavy chain variable region comprising SEQ ID NO:
 2. 5. A methodof treating a cocaine-related disorder in an individual, comprisingadministering to the individual a therapeutic amount of a monoclonalantibody that specifically binds cocaine, wherein the antibodycomprises: (a) a murine lambda light chain variable region CDR1comprising SEQ ID NO: 4; (b) a murine lambda light chain variable regionCDR2 comprising SEQ ID NO: 5; (c) a murine lambda light chain variableregion CDR3 comprising SEQ ID NO: 6; (d) a human gamma heavy chainvariable region CDR1 comprising SEQ ID NO: 7; (e) a human gamma heavychain variable region CDR2 comprising SEQ ID NO: 8; (f) a human gammaheavy chain variable region CDR3 comprising SEQ ID NO: 9; and (g) ahuman light chain constant region.
 6. The method of claim 5, wherein thehuman light chain constant region is selected from the group consistingof a human kappa light chain constant region and a human lambda lightchain constant region.
 7. The method of claim 6, wherein the human lightchain constant region is a human lambda light chain constant regioncomprising SEQ ID NO:
 22. 8. The method of claim 5, wherein the antibodyhas a murine lambda light chain variable region comprising SEQ ID NO: 1and a human gamma heavy chain variable region comprising SEQ ID NO: 2.